The mammary gland is a compound tubulo-alveolar gland that is composed of a series of branched ducts that, during lactation, drain sac-like alveoli (lobules). In the rodents, the mammary epithelium is embedded within a mammary fat pad, whereas in humans, it is embedded within a fibrous and fatty connective tissue. The mammary epithelium is composed of two lineages of epithelial cells: the luminal cells (which make milk during lactation) and basal positioned myoepithelial cells. Like other epithelia, the mammary gland is organized into undifferentiated stem cells and the majority, differentiated cells. Currently, there is no single cell surface biomarker available that allows substantial enrichment of somatic mammary stem cells. All known enrichment protocols rely on combinations of cell surface markers.
While the somatic mammary stem cells (and possibly some of their more immediate descendants that have decreased stem cell potential but still have proliferative potential) may be the targets for malignant transformation, mammary malignancies themselves have been shown to have a cancer stem cell component that propagates the tumor (Al-Hajj M et al. Proc Natl Acad Sci USA 2003,100:3983-8). The presence of tumor stem cells provides an explanation as to why some treatments seem to be effective initially but tumors recur later. Treatments that attack the differentiated tumor cells may not affect the small population of tumor stem cells that actually give rise to tumors. Thus, it is important that the tumor stem cell population be targeted in order for tumors to be successfully contained or eradicated. Cell surface markers for mammary tumor stem cells are of great value in this regard.
Wnt proteins are a family of highly conserved secreted growth factors. Wnt proteins are divided into two types: canonical and noncanonical, and activate different downstream signal transduction pathways. Wnt proteins that are classified as canonical include, but are not limited to Wnt1, Wnt2, Wnt3, Wnt3a, and Wnt8 (Liu G et al. Mol Cell Biol 2005, 25:3475-3482). In the canonical pathway, a Wnt protein initiates signals by binding to a protein complex containing a member of the Frizzled family of seven-transmembrane-domain receptors and a molecule with homology to the low density lipoprotein (LDL) receptor (LRP5 and LRP6) (Logan C Y and R Nusse, Ann Rev Cell Dev Biol 2004, 20:781-810). This down regulates glycogen synthase kinase-3 (GSK3) activity. Normally, GSK3 phosphorylates β-catenin, marking it for ubiquitin-dependent degradation. Thus, GSK3 inhibition results in increased β-catenin levels in the cytosol and nucleus, allowing physical interaction of the Tcf/Lef class of DNA-binding proteins and activation of target promoters (Logan C Y and R Nusse, Ann Rev Cell Dev Biol 2004, 20:781-810).
In addition, other proteins regulate the activity of the Wnt pathway at several levels. Secreted Frizzled-related proteins, Norrin, Dickkopf (DKK), Wise, connective tissue growth factor, and Kremen regulate signaling at the level of the Wnt/Frizzled/LRP interaction while other proteins, including APC, control the pathway intracellularly (Finch P W et al., Proc Natl Acad Sci USA 1997, 94:6770-6775; Wang S M et al. 1997, Biochem Biophys Res Commun 1997, 236:502-504; Xu Q et al. Cell 2004, 116:883-895; Semenov M V et al. Curr Biol 2001, 11:951-961; Mao B et al. Nature 2001, 411:321-325; Itasaki N et al. Development 2003, 130:4295-4305; Mercurio S et al. Development 2004, 131-2137-2147; and Mao B et al. Nature 2002, 417:664-667). The Dickkopf (DKK) family of secreted proteins are antagonists of the canonical Wnt pathway (Bafico A et al. Nat Cell Biol 2001, 3:683-686; Mao B et al. Nature 2001, 411:321-325). Whereas Wnt-Frizzled interactions may also be involved in non-canonical Wnt signaling events, the LRP5/6 moiety appears to be specifically required for the canonical pathway (Liu G et al. Mol Cell Biol 2005, 25:3475-3482).
Published studies suggest that canonical Wnt signaling plays a significant role during normal mammary gland development (Andl T et al. Dev Cell 2002, 2:643-653; Brisken C et al. Genes Dev 2000, 14:650-654; Hsu W et al. J Cell Biol 2001, 155:1055-1064; Tepera S B et al. J Cell Sci 2003, 116:1137-1149; and van Genderen C et al. Genes Dev 1994, 8:2691-2703). The normal mammary gland development in mice begins at approximately embryonic day 10.5 with the formation of two “mammary lines” (Veltmatt J M et al. Differentiation 2003, 71:1-17). In response to signals from the underlying mesenchyme, the mammary lines give rise to five pairs of lens-shaped mammary placodes which subsequently transform into buds of epithelial cells and sink into dermis. Activation of the canonical Wnt pathway along the mammary lines coincides with the initiation of mammary morphogenesis, and subsequently localizes to mammary placodes and buds (Chu E Y et al. Development 2004, 131:4819-4829). Several Wnt ligands and receptor genes, including LRP5, are expressed during embryonic mammary morphogenesis (Chu E Y et al. Development 2004, 131:4819-4829). Embryos expressing the canonical Wnt inhibitor DKK1 display a complete block in the formation of mammary placodes and mice deficient of Lef-1 fail to maintain their mammary buds (Andl T et al. Dev Cell 2002, 2:643-653; and van Genderen C et al. Genes Dev 1994, 8:2691-2703). DKK1 inhibits the Wnt signaling pathway by binding to LRP5 and LRP6 (Bafico A et al. Nat Cell Biol 2001, 3:683-686).
A connection between mammary stem cells and Wnt1- or β-catenin-induced tumorigenesis has been established. Transgenic expression of these genes result in widespread mammary hyperplasia and rapid tumor formation (Imbert A et al. J Cell Biol 2001, 153:555-568; Nusse R and Varmus H E Cell 1982, 31:99-109; and Tsukamoto A S et al. Cell 1988, 55:619-625). The hyperplastic tissue contains an increased ratio of mammary stem cells which are thought to directly give rise to transformed cells (Li Y et al. Proc Natl Acad Sci USA 2003, 100:15853-15858; Liu B et al. Proc Natl Acad Sci USA 2004, 101:4158-4163; and Shackleton M et al. Nature 2006, 439:84-88). Tumors arising from stem cells often show mixed lineage differentiation (Owens D M and Watt F M Nat Rev Cancer 2003, 3:444-451) and tumors induced by Wnt effectors indeed contain cells from both epithelial lineages (Li Y et al. Proc Natl Acad Sci USA 2003, 100:15853-15858; Liu B et al. Proc Natl Acad Sci USA 2004, 101:4158-4163; and Rosner A et al. Am J Path 2002, 161:1087-1097). The large majority of human breast tumors overexpress cytoplasmic and nuclear levels of β-catenin, a hallmark of activation of the canonical Wnt pathway (Lin S Y et al. Proc Natl Acad Sci USA 2000, 97:4262-4266; and Ryo A et al. Nat Cell Biol 2001, 3:793-801). In addition, many human breast tumors up-regulate Pin1, which inhibits β-catenin degradation by preventing its association with APC (Ryo A et al. Nat Cell Biol 2001, 3:793-801; and Wulf G M et al. EMBO J 2001, 20:3459-3472). Another recent report links amplification and overexpression of Dishevelled1, a positively acting component of the pathway upstream of GSK3, to breast cancer (Nagahata T et al. Cancer Sci 2003, 94:515-518). Further, recent reports have linked down-regulation of the secreted Wnt inhibitors sFRP1 and Wif1 to breast cancer (Ugolini F et al. Oncogene 2001, 20:5810-5817; Klopocki E et al. Int J Oncol 2004, 25:641-649; and Wismann C et al. J Pathol 2003, 201:204-212).